Comparisons of a species’ genetic diversity and divergence patterns across large connected populations versus isolated relict areas provide important data for understanding potential response to global warming, habitat alterations, and other perturbations. Aquatic taxa offer ideal case studies for interpreting these patterns, because their dispersal and gene flow often are constrained through narrow connectivity channels that have changed over geological time and/or from contemporary anthropogenic perturbations. Our research objective is to better understand the interplay between historic influences and modern-day factors (fishery exploitation, stocking supplementation, and habitat loss) in shaping population genetic patterns of the yellow perch Perca flavescens (Percidae: Teleostei) across its native North American range. We employ a modified landscape genetics approach, analyzing sequences from the entire mitochondrial DNA control region (912 base pairs) and 15 nuclear DNA microsatellite loci of 664 spawning adults from 24 populations. Results support that perch from three primary glacial refugium areas (Missourian, Mississippian, and Atlantic) founded contemporary northern populations. Genetic diversity today is highest in southern (never glaciated) populations, and also is appreciable in northern areas that were founded from multiple refugia. Divergence is greater among isolated populations, both north and south; the southern Gulf Coast relict populations are the most divergent, reflecting their long history of isolation. Understanding the influence of past and current waterway connections on the genetic structure of yellow perch populations may help us to assess the roles of ongoing climate change and habitat disruptions towards conserving aquatic biodiversity.